Real-time PCR enables continuous monitoring of labels during the generation of PCR products. Currently, available methods utilize either labeled probes or DNA intercalating dye to monitor the amplification of PCR products. For this purpose, single oligonucleotide probes containing a fluorophore and a quencher placed e.g. 10-30 bases apart may be used. Such a TaqMan probe can be hydrolyzed during the amplification due to the 5′-3′ double strand-specific exonuclease activity of a Taq polymerase that separates the fluorophore from the quencher, resulting in fluorescence increase. Real-time PCR instruments are typically used which are equipped with fluorescence detectors and software capable of estimating the cycle threshold (Ct), which is the cycle at which fluorescence is greater than background fluorescence, for positive reactions.
However, well recognized difficulties in reproducing published tests due to variation in performance of PCR thermocyclers, inefficiency of different DNA polymerases, personnel and the presence of PCR inhibitors in the sample matrix can hamper implementation in laboratories, particularly those with extensive quality assurance programs. Lack of reproducible methods often forces testing laboratories to spend substantial resources on adaptation of the published tests. It is thus necessary to have internationally validated, open-formula PCR-based methods available in which the target gene, performance characteristics and validation criteria are known and which follow the ISO criteria for validation of alternative microbiological methods.
A major drawback of some published PCRs is that they do not contain an internal amplification control. In contrast to a (external) positive control, an internal amplification control is a non-target DNA sequence present in the very same sample, which is co-amplified simultaneously with the target sequence. In a PCR without an internal amplification control, a negative response (no band or signal) could mean that there was no target sequence present in the reaction. But, it could also mean that the reaction was inhibited, due to malfunction of thermal cycler, incorrect PCR mixture, poor DNA polymerase activity, or not least the presence of inhibitory substances in the sample matrix. In particular, after nucleic acid extraction, inhibitors may still be present from clinical samples (e.g. hemoglobin), environmental samples (e.g. humic and fulvic acids), and chemicals employed during nucleic acid extraction (e.g. ethanol detergents, or chaotropic agents). It is desirable to differentiate a true negative result from a false negative when PCR is affected by amplification inhibitors.
The reliability of diagnostic assays can be increased by the inclusion of an internal control nucleic acid that can indicate the presence and impact of PCR inhibitors. An internal positive control is usually amplified simultaneously in the presence of a target sequence using a labeled fluorophore that emits light at a different wavelength than the fluorophore used for the target sequence assay, with the two fluorophores detected in different channels by the real-time PCR instrument. The commonly used internal controls for PCR are plasmids that contain a sequence similar to that of the assay target except for probe region. A limited number of internal positive control molecules may be added to individual assay target and co-amplified with target nucleic acid. Thus, an internal positive control signal is evidence that the amplification reaction proceeded sufficiently to generate a positive signal from very small quantities of target nucleic acid.
However, some devices such as the LightCycler 1.2 and LightCycler 2 (Roche Applied Science, Indianapolis, Ind., USA), the Ruggedized Advanced Pathogen Identification Device (R.A.P.I.D.) instrument (Idaho Technology, Salt Lake City, Utah, USA), and handheld real-time PCR instruments are only equipped with one light source and associated emission channel.
To overcome this deficiency, P. Jothikumar et al., Biotechniques 46: 519-524 (2009) developed a design to create a triple-labeled probe as an internal positive control (IPC) that utilizes a combination of the fluorescence resonance energy transfer (FRET) and TaqMan techniques. The IPC probe, labeled with FAM and Cy5.5 fluorophores at the 5′ end and Black Hole Quencher (BHQ) at the 3′ end, enabled Cy5.5 emission through energy transfer from the FAM fluorophore. The second, target-specific TaqMan assay in the multiplex used an FAM and BHQ1-labeled probe at the 5′ and 3′ ends, respectively. Thus, one excitation source was used to generate two different fluorescence emissions (FAM and Cy5,5) that were measured in two separate channels by the real-time PCR instrument.
Rosenstraus et al., Journal of Clinical Microbiology, 36, 191-197 (1998) describe internal controls for routine diagnostic PCR, wherein the specific target and the internal control were detected in separate reactions using separate, target- and internal control-specific, oligonucleotide capture probes.
Gruber et al., Applied and Environmental Microbiology, 67, 2837-2839 (2001) describe a real-time PCR method to quantitate viral DNA that includes duplex amplification, internal standardization, and two-color fluorescence detection without the need to generate an external standardization curve.
However, there is still a need in the art for suitable methods for detecting multiple nucleic acids, e.g. target nucleic acids and internal control nucleic acids, in a sample.